ATCC persistent infection. In addition, LAMA5 and HSPG2 were downregulated during CVB1 10796 persistent infection and remained unchanged in CVB1 ATCC persistent infection. ALDH1A3 is used as a marker of dedifferentiated pancreatic β- cells (250,288) and SUSD2 as a marker of a particular stage in the maturation of the endocrine pancreas during fetal pancreatic differentiation (252). Interestingly, when upregulated during CVB1 persistent infection, SPARC is secreted by the endocrine and exocrine pancreas and it may have a negative impact on β-cell growth and survival (251). HSPG2 and LAMA5, downregulated by CVB1 10796 persistent infection, are components of the human peri-islet basement membrane, which protects pancreatic islets from immune cell infiltration and thereby from β-cell killing (253,254). Downregulation of the HSPG2 and LAMA5 in CVB1 persistent infection may make pancreatic islets more susceptible for leukocyte infiltration. In conclusion, the observed upregulation of SPARC and downregulation of HSPG2 and LAMA5 in persistently infected cells suggests that persistent CVB infection in β-cells may decrease the viability and function of these cells.
6.3 The accumulation of mutations in CVB1 genome during viral
had mutations in the puff region and six of the PIDVs had mutations in the knob region. Additionally, five of the PIDVs had mutations in the infection-enhancing epitope of VP4 (AA11-30). The VP2 puff region and VP3 knob region include neutralizing immunogenic sites and contribute to the receptor binding sites in polioviruses (294,295) and rhinoviruses (296,297). Furthermore, mutations in these regions have been shown to modulate the virulence and ability of CVBs to induce myocarditis in mice (298-300). In addition, the regulation of plaque morphology of CVB3 is associated with a single AA substation in the VP3 knob region (301).
Another mutation in AA16 (G16R) at the infection-enhancing epitope region of CVB4 VP4 is associated with the plaque phenotype, with in vitro avirulence, and the in vivo intermediate phenotype in mice (302,303). Among the five PIDVs having mutations in the infection-enhancing epitope in VP4, the ATCC E2-PIDV[PANC-1]
had the mutation G16R. The carrier state type of persistent CVB4 infection in PANC-1 cells resulted in non-synonymous mutations in VP1 and VP2 structural proteins, and in 2A, 2C and 3D non-structural proteins (37). The majority of these observed mutations were located at VP1 and 2A proteins, and none in the CAR footprint region (37). Also, in this study, only three AAs in the CAR footprint region were identified to have mutations, AA202 was mutated in three of the eight PIDVs, AA138 in one of the PIDVs with a mutation and in AA165, three of the PIDVS had mutation.
In VP1 the most prominent exterior loop is the DE loop, which is located at the fivefold axis symmetry of the virus capsid (259). Furthermore, because of the icosahedral symmetry, the DE loops of neighboring subunits interact with each other. The interaction may stabilize the capsid, mediate pH stability of the virus since the putative ion-binding site is along the fivefold axis of symmetry. Therefore, the mutations in the DE loop region where all the PIDVs had mutations may interfere with the capsid and modulate the pH stability of the virus. However, we do not have data to support this hypothesis. Furthermore, the majority of the PIDVs had mutations on the BC loop region, which flanks the rim of the canyon closest to the fivefold axes of symmetry and has shown to contain neutralizing immunogenic sites of EVs, including polioviruses (294,295), rhinoviruses (296,297) and coxsackieviruses (CVB4) (304).
Previously, it was considered that EVs have single ORF, but the discovery of a second ORF from human EVs (Enteroviruses A, B and C) overturned the impression (14,15). Interestingly, the sequence, of a recently discovered second ORF2, which translated in to a second ORF protein ORF2p, was identified from the current CVB1 sequences. The ORF2p is a membrane-associated protein that is localized to
cytosolic vesicles, which seems to facilitate intestinal infection and the release of viral particles from intestinal epithelial cells (14). It contains a highly conserved WIGHP domain, having an important function in ORF2p-dependent viral intestinal infection (14,15). The WIGPH domains were recognized from PIDV sequences, and has remained conserved, except for the 10802-PIDV[1.1B4], which contained one mutation, V18A, in the region. Therefore, it is unlikely that this ORF would play an important role in the development of CVB persistency in cell model.
The 5′ UTR has domains I-VI and domain I is known as the cloverleaf region, which plays a role in viral RNA replication (45,305). Domains I-V have the internal ribosome entry site (IRES), functioning in viral mRNA translation (306). The read depth in publication II analyses remained below 10 in PIDV genomes in the cloverleaf region, leading to variant calling not being possible. The 40S ribosomal subunit binds to a polypyrimidine tract in IRES (233) and the mutations at the IRES may influence the virulence. For example, a site-directed mutagenesis showed that mutation at CVB1, nt 573 and 579 within the 5′-UTR in CVB1 genome leads to elevated virulence and pathogenesis of the virus (307). Additionally, a proper folding and functioning of the 5’-UTR may influence viral replication efficiency and virulence (308). Publication II identified several mutations in 5’-UTR, however, the mutations were not all mapped to highly reactive base pairs, except both 10802- PIDV[PANC-1] and [1.1B4], which had mutations in the base pair 234. This is categorized as highly reactive (308) being more sensitive for chemical modification in solution.
The reactivity of the base pair depends on ribose backbone flexibility, which is determined by base pairing and other conformational constraints. The read depth in the 3’-UTR remained mainly below, ruling out the area from further analyses. In summary, the observed mutations may influence the EV pathogenesis in several ways and besides the effect of single mutations, also combinations of different mutations in certain functional areas may lead to changes in the viral genome supporting the persistent infection.
6.3.1 The K257R mutation identified from all persistent infection derived viruses
One specific mutation of the VP1 structural protein, K257R, was identified from all the analyzed PIDVs. Therefore, this mutation can be considered as a hallmark of CVB persistency. The location of the mutation is close to the C-terminus of VP1, and it is near the amino acid K259, which is the known CAR binding site. The
mutation also locates at the published site of CVA16 immunogenic peptide (PEP91) (309). The mutation has also been reported previously by Smura et. al. (310). The mutation was formed after blind-passaging the CVB6 Smith strain, which naturally causes non-lytic infection in human pancreatic duct epithelial HPDE cells. The virus CVB6 Smith strain turned from non-lytic into lytic form suggesting that the AA257 may play a role in the virus life cycle. It was also suggested that it influences the DAF binding phenotype, however, the effect was minor (310). In addition, the DAF is not detected by proteomics from cell lysates of CVB1 ATCC and 10796 persistently infected PANC-1 cells as shown in publication I by proteomics, suggesting the persisting CVB strains hardly use DAF as a receptor. The appearance of the mutations in all eight studied PIDVs makes it highly interesting for further studies.
Such studies could include side-directed mutagenesis to vary this AA and study its effect on the course of infection in cell and animal models.